Zygonemella: the forgotten genus of the family Xyalidae (Nematoda)

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1 Universidade de São Paulo Biblioteca Digital da Produção Intelectual - BDPI Centro de Biologia Marinha - CEBIMar Artigos e Materiais de Revistas Científicas - CEBIMar Zygonemella: the forgotten genus of the family Xyalidae (Nematoda) Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

2 Zootaxa 3669 (2): Copyright 2013 Magnolia Press Correspondence Zygonemella: the forgotten genus of the family Xyalidae (Nematoda) ISSN (print edition) ZOOTAXA ISSN (online edition) BEATRIZ PEREIRA CUNHA 1, SIMONE BRITO & GUSTAVO FONSECA Centro de Biologia Marinha da Universidade de São Paulo, Rod. Manoel Hyppólito do Rego km 131,5, São Sebastião,São Paulo, Brazil 1 Corresponding author. beatriz.pecunha@gmail.com Key words: nematode, species description, synonym, 18S, morphometry Species descriptions of the family Xyalidae, as well as of most marine nematodes, were in general largely made in the past century (e.g. Allgén, 1927; Cobb, 1920; Gerlach, 1957; Lorenzen, 1977). Many of these descriptions were based on one or two specimens or even on juveniles with relatively few features of taxonomic value. Lack of types and inaccuracy in sampling localities are other problems associated with taxonomy of marine nematodes. These issues, together with the fact that in the past researchers had slower exchange of information and reduced access to some journals, led to the multiplication of synonyms. To propose a new species of nematode, particularly within a genus with a convoluted historical background, requires caution and critical taxonomical review prior to the description (Adams, 2001; Fonseca & Decraemer, 2008). Nematodes species delimitation based solely on morphological characters is problematic for three reasons: a) high phenotypic plasticity among populations (Sommer and Ogawa, 2011), which reduces the number of diagnostic characters; b) poor taxonomical descriptions (Nadler, 2002); and c) existence of cryptic species (Derycke et al., 2005; Fonseca et al., 2008). These problems often lead to greater taxonomic uncertainty within very speciose genera and may bias diversity studies. This problem is particularly evident in the species-rich and often ecologically dominant Xyalidae Chitwood, This family contains 44 genera, with some genera, such as Daptonema Cobb, 1920 and Theristus Bastian, 1865, having more than one hundred nominal species (Fonseca & Bezerra, 2012). Most species descriptions are limited to a few and poorly described diagnostic morphological characters. Moreover, identification keys and systematic revisions (e.g. Lorenzen, 1977; Fonseca & Bezerra, 2012) need to be revised and updated. Daptonema matrona Neres, Fonseca-Genevois, Torres, Cavalcanti, Castro, Da Silva, Rieger, & Decraemer 2010 was recently described from Pina Basin, an estuarine area located on the coast of the state of Pernambuco (Brazil). The main diagnostic characters given by the authors were: 1) reduced cephalic setae in relation to the head diameter, 2) straight shape of the spicules, 3) amphidial fovea slightly oval, situated less than one head diameter from the anterior end and 4) viviparous reproduction. To accommodate this new species, the authors amended the diagnosis of the genus Daptonema proposed by Lorenzen (1977). According to Lorenzen, Daptonema species had L-shaped spicules and four cephalic setae longer than 5µm in length. The spicules of D. matrona are straight, and their cephalic setae are smaller than 5µm (Neres et al., 2010). However, the four characters listed by Neres et al. (2010) are also the diagnostic characters of the genus Zygonemella erected by Cobb (1920) from material sampled in Punta Arenas, Pacific coast of Costa Rica. Gerlach (1957) identified some specimens of Z. striata from the mangrove of Cananéia, south coast of São Paulo, Brazil. In his work, Gerlach pointed out that although the specimen from Costa Rica had a larger b-ratio (rate of body length divided by the pharynx length) and tail length than specimens from Cananéia, he considered these differences insufficient to erect a new species. Further revisions recognized the validity of Zygonemella as a distinct genus from Daptonema (Lorenzen, 1977; Gerlach and Riemann, 1974; Nicholas & Trueman, 2002; Fonseca & Bezerra, 2012). When comparing the descriptions of Z. striata and D. matrona, the only two morphological differences reported are the presence of a gubernaculum and the presence of five ejaculatory glands in the specimens examined by Neres et al. (2010). The gubernaculum was neither mentioned by Cobb or Gerlach, and the male described by Cobb had 10 ejaculatory glands. To determine if D. matrona should be synonymized with Z. striata, we sampled specimens from the coast of São Paulo (mangroves at Ubatumirim, Guaratuba, Juréia-Itatins and Cananéia) including the sampling localities previously sampled by Gerlach (1957) at Cananéia and compared with the published data by Neres et al. (2010) from Pernambuco. Accepted by Kerrie Davies: 2 May 2013; published: 6 Jun Licensed under a Creative Commons Attribution License 179

3 The material was fixed with DESS (Yoder et al., 2006) and stored at room temperature. One specimen from Ubatumirim mangrove was picked for molecular analyses. DNA extraction was done with Worm Lysis Buffer according to Derycke et al. (2005) and the primer set G18S-18P was used to amplify the small ribosomal subunit (18S rdna) according to Fonseca & Fehrlauer-Ale (2012). Complementary strands were combined, edited and compiled using Geneious Pro v5.6.5 created by Biomatters (Available from When compared with the sequence from Pernambuco obtained by Neres et al. (2010), our sequence (GenBank KC920423) showed only four nucleotide bases of difference, representing 99.8% similarity between the two sequences. This congruence supports the idea that specimens from Pernambuco and São Paulo belongs to the same species (Bhaduri et al., 2006). For morphological comparisons, 24 specimens from São Paulo coast were measured (Table 1 and 2). Specimens of Z. striata from all four localities (Costa Rica, Cobb 1920; Cananéia, Gerlach 1957; Pernambuco, Neres et al. 2010; São Paulo present study) were analyzed by means of cluster analysis calculated on a Euclidean matrix using the group linkage algorithm and associated SIMPROF procedure (Clarke & Gorley, 2006). Prior to the analysis characters were standardized and highly correlated characters (r>0.8) were excluded. TABLE 1. Females body measurements of Zygonemella striata [means and (range) in µm]. Measurements available to only one location were excluded from the table. n, number of specimens measured; a, body length divided by maximum body diameter; b, body length divided by pharynx length; c, body length divided by tail length; c, tail length divided by anal body diameter; L, body length; mbd, maximum body diameter; ph, pharynx length; ph bd, pharynx base diameter; t, tail length ; abd, anal body diameter; b.cav, buccal cavity length ; hd, head diameter; n. ring, position of nerve ring from anterior body end; n. ringbd, body diameter in nerve ring region; Amph%, percentage of diameter amphidial fovea in relation to corresponding body diameter; amphd pos, distance of amphidial fovea from anterior end; els, external labial setae length; cs, cephalic setae length; ts, caudal setae length; V%, position of the vulva as percentage of body length from anterior end; V, position of vulva from anterior body end; vbd, body diameter in vulva. Pernambuco São Paulo Cananéia-São Paulo (Neres et al. 2010) (present study) (Gerlach, 1957) n L ( ) ( ) 1480 ph ( ) ( ) 250 mbd 88.4 ( ) 61.3 ( ) 125 t ( ) 170 ( ) 280 a 14.7 ( ) 14.9 ( ) 12 b 6.4 (6 6.9) 5.8 (5 6.6) 5.9 c 5.89 ( ) 5.4 ( ) 5.3 c` 3.9 ( ) 5 (3.8 6) 2.9 V% 74.2 ( ) 71 (64 76) 70 els 3.4 ( ) 3.7 (2 5.6) 3.5 cs 2 ( ) 2 ( ) - ph bd 75.4 ( ) 54.4 ( ) 108 b.cav 12.5 (9 14.4) 10.1 (7 15.7) - hd 31.8 (30 36) 17.6 ( ) 38 n.ring 98.5 ( ) n.ringbd 63.4 ( ) - 95 Amph% 11.2 ( ) 14 (7.4 19) - amph pos 14.3 ( ) 13.3 (10 20) 5 abd 55.7 ( ) 34.8 ( ) 95 V ( ) ( ) 1040 vbd 75.2 ( ) ts 8.2 ( ) 6.7 ( ) Zootaxa 3669 (2) 2013 Magnolia Press CUNHA ET AL.

4 TABLE 2. Male s body measurements of Zygonemella striata [means and (range) in µm]. Measurements available to only one location were excluded from the table. n, number of specimens measured; a, body length divided by maximum body diameter; b, body length divided by pharynx length; c, body length divided by tail length; c, tail length divided by anal body diameter; L, body length; mbd, maximum body diameter; ph, pharynx length; ph bd, pharynx base diameter; t, tail length; abd, anal body diameter; b.cav, buccal cavity length ; hd, head diameter; n. ring, position of nerve ring from anterior body end; n. ringbd, body diameter in nerve ring region; Amph%, percentage of diameter amphidial fovea in relation to corresponding body diameter; amphd pos, distance of amphidial fovea from anterior end; els, external labial setae length; cs, cephalic setae length; ts, caudal setae length; spic, spicule length (along the spicule); gub, gubernaculum length. Pernambuco São Paulo Cananéia-São Paulo Costa Rica (Neres et al., 2010) (present study) (Gerlach, 1957) (Cobb, 1920) n L ( ) ( ) ph ( ) ( ) mbd 64.4 ( ) 46.4 ( ) t 192 ( ) ( ) a 18.8 ( ) 18.6 ( ) b 6.2 (6-6.6) 6.1 ( ) c 6.5 (6-6.8) 6.4 ( ) c` 4.4 ( ) 4.4 (3.7-5) els 3.1 (3-3.6) 4.1 ( ) cs ( ) - - ph bd 58.9 ( ) 41.6 ( ) b.cav 11.8 ( ) 8.4 ( ) - - hd 24.7 ( ) 16.8 ( ) n.ring 92.9 ( ) 71.7 ( ) n.ringbd 49.4 ( ) 40 ( ) Amph% 20 ( ) 20 (16-26) - - amph pos 12.8 ( ) 10.6 ( ) abd 44.1 ( ) 30.4 ( ) spic 30.3 ( ) 23 ( ) 30 - gub 6.6 ( ) 5.5 ( ) - - sipc/abd ts 8.6 ( ) 6.2 (4.6-8) - - Specimens from the four localities are morphologically similar (Fig. 1) with considerable overlap between morphometric characters (Table 1, 2). The only exceptions are: (1) the females from Pernambuco are longer than specimens from the present study (Table 1); (2) the males from Pernambuco are slightly longer (365 µm on average) and with longer pharynx (55 µm on average) than males from the present study (Table 2). When the specimens from Pernambuco are compared with those described by Gerlach, also from Cananéia, the only character that is different is the total body length of the males (a difference of 249 µm), the others are within the range reported by Neres et al. (2010). Apart from body length, which can vary within a population according to food availability (Herman and Vranken, 1988), the measurements do not give enough support to distinguish the specimens from the four localities. Morphometric analysis of the females confirmed no clear clustering of the specimens from the different locations (Figure 2a). Female specimens from Pernambuco are clustered with the specimens from São Paulo (present study). Analysis using the males also showed no separation between locations (Figure 2b). Multivariate analysis indicated that although these specimens are several hundreds of kilometers apart from each other, they are morphologically similar. Molecular and morphometric data suggest that these specimens are probably from one single species broadly spread along the Brazilian coast up to Costa Rica. So far, this species has been reported from estuarine areas with mangrove ZYGONEMELLA: THE FORGOTTEN GENUS OF THE FAMILY XYALIDAE Zootaxa 3669 (2) 2013 Magnolia Press 181

5 forest. It is important to note, however, that our conclusions are based on evidence from the ribosomal gene 18S-DNA. This is a conservative gene unsuitable for detecting population level structuring in nematodes (Bhaduri et al., 2006). Additional information could be gained if a more variable molecular marker, such as mitochondrial COI, was used. At the moment, we cannot exclude the possibility that these populations are in fact a complex of cryptic species without morphological differences (Derycke et al., 2008; Fonseca et al., 2008). FIGURE 1. Anterior end of the males from the four localities where Zygonemalla striata has been reported. A) Costa Rica (adapted from Cobb, 1920); B) Cananéia São Paulo (adapted from Gerlach,1957); C) Pernambuco (adapted from Neres et al., 2010); D) São Paulo (present study). Depicted spicules and gubernaculum from Pernambuco (E; adapted from Neres et al., 2010) and São Paulo (F; present study). FIGURE 2. Dendogram based on the morphometrics of Zygonemella striata specimens from the four locations. Dashed lines represent specimens with no significant difference (p > 0.5; SIMPROF). a) females; b) males. : São Paulo (present study); : Pernambuco (Neres et al., 2010); : Cananéia - São Paulo (Gerlach, 1957); X: Costa Rica (Cobb, 1920). In addition to the overlapping of characters between populations, another feature in common among specimens from São Paulo and the Pernambuco is the presence of a small gubernaculum without apophysis at the tip of the spicules (Figure 1E, F). Such a character is difficult to observe and might have been overlooked by Cobb (1920) and Gerlach (1957). Moreover, the material deposited in the National Museum of Rio de Janeiro by Neres et al. (2010) shows the presence of ten ejaculatory glands, five on each side of the intestine, in agreement with the description made by Cobb and the specimens analyzed in the present study. There is so far no strong evidence to separate D. matrona from Z. striata, 182 Zootaxa 3669 (2) 2013 Magnolia Press CUNHA ET AL.

6 and they should be considered synonyms. This study reinforces the importance of critically reviewing the literature before describing new species, especially when the family in question has several species with poorly defined morphological characters. Acknowledgments We thank two anonymous reviewers for their critical reading and suggestions, and Dr. Rick Hochberg for proof reading our manuscript. This study was financed by FAPESP 2009/ This is a publication of the NP-Biomar/USP. References Adams, B. (2001) The species delimitation uncertainty principle. Journal of Nematology, 33, Allgén, C. (1927) Freilebende marine nematoden von der Campbell- und Stateniseln. Nyt Magazin Naturvidenskaberne, 66, Bhadury, P., Austen, M.C., Bilton, D.T., Lambshead, P.J.D., Rogers, A.D. & Smerdon, G.R. (2006) Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes. Marine Ecology Progress Series, 320, 1 9. Clarke, K.R. & Gorley, R.N. (2006) PRIMER v6: User Manual/Tutorial. PRIMER-E Ltd, Plymouth, UK. Cobb, N.A. (1920) One hundred new nemas (type species of 100 new genera). Contributions to a Science of Nematology, 9, Derycke, S., Remerie, T., Vierstraete, A., Backeljau, T., Vanfleteren, J., Vincx, M. & Moens, T. (2005) Mitochondrial DNA variation and cryptic speciation within the free-living marine nematode Pellioditis marina. Marine Ecology Progress Series, 300, Derycke S., Remerie T., Backeljau T., Vierstraete A., Vanfleteren J., Vincx M. & Moens T. (2008) Phylogeography of the Rhabditis (Pellioditis) marina species complex: evidence for long-distance dispersal, and for range expansions and restricted gene flow in the northeast Atlantic. Molecular Ecology, 17, Fonseca, G. & Decraemer, W. (2008) State of the art of the free-living marine Monhysteridae (Nematoda). Journal of the Marine Biological Association of the United Kingdom, 88, Fonseca, G., Derycke, S. & Moens, T. (2008) Integrative taxonomy in two free-living nematode species complexes. Biological Journal of the Linnean Society, 94, Fonseca, G. & Fehlauer-Ale, K.H. (2012) Three in one: fixing marine nematodes for ecological, molecular and morphological studies. Limnology and Oceanography: Methods, 10, Fonseca, G. & Bezerra, T.N. (2012) Order Monhysterida Filipjev, Zoology Online. Germany: De Gruyter. Gerlach, S.A. (1957) Marine Nematoden aus dem Mangrove-Gebiet von Cananeia (Brasilianische Meeres-Nematoden III). Jahrbuch Akademie der Wissenschaften und der Literatur in Mainz, 5, Gerlach, S.A. & Riemann, F. (1974) The Bremerhaven Checklist of Aquatic Nematodes: A Catalogue of Nematoda Adenophorea Excluding the Dorylaimida. Part 2. Veröffentlichungen des Instituts für Meeresforschung in Bremerhaven, 4 (Supplement), Herman, P.M.J. & Vranken, G. (1988) Studies of the life-history and energetics of marine and brackish-water nematodes II. Production, respiration and food uptake by Monhystera disjuncta. Oecologia, 77, Lorenzen, S. (1977) Revision der Xyalidae (freilebende Nematoden) auf der Grundlage einer kritischen Analyse von 56 Arten aus Nord-und Ostsee. Veröffentlichungen des Institut für Meeresforschungen Bremerhaven, 16, Nadler, S.A. (2002) Species delimitation and nematode biodiversity: phylogenies rule. Nematology, 4, Neres, P.F., Fonseca-Genevois, V.G., Torres, R.A. Cavalcanti, M.F., Castro, F.J.V., Da Silva, N.R.R, Rieger, T.T. & Decraemer, W. (2010) Morphological and molecular taxonomy of a new Daptonema (Nematoda, Xyalidae) with comments on the systematics of some related taxa. Zoological Journal of the Linnean Society, 158, Nicholas, W.L. & Trueman, J.W.H. (2002) The taxonomy of the family Xyalidae Chitwood, 1951 (Monhysterida: Nematoda): a cladistic analysis. Nematology, 4, Sommer, R.J. & Ogawa, A. (2011) Hormone Signaling and Phenotypic Plasticity in Nematode Development and Evolution. Current Biology, 21, Yoder, M., De Ley, I.T., King, I.W., Mundo-Ocampo, M., Mann, J., Blaxter, M., Poiras, L. & De Ley, P. (2006) DESS: A versatile solution for preserving morphology and extractable DNA of nematodes. Nematology, 8, ZYGONEMELLA: THE FORGOTTEN GENUS OF THE FAMILY XYALIDAE Zootaxa 3669 (2) 2013 Magnolia Press 183